Galanin receptor GALR3 and nucleotides encoding same

ABSTRACT

A new galanin receptor, GALR3, is described. Also provided are nucleic acids encoding same and various assays to identify ligands particular to said receptor. Ligands so identified are useful for the treatment of obesity, treatment of pain, and treatment of cognitive disorders.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Not applicable

STATEMENT REGARDING FEDERALLY-SPONSORED R&D

[0002] Not applicable

REFERENCE TO MICROFICHE APPENDIX

[0003] Not applicable

FIELD OF THE INVENTION

[0004] This invention relates to a novel galanin receptor, designated GALR3, to nucleotides encoding same, and to assays making use thereof.

BACKGROUND OF THE INVENTION

[0005] Although first isolated from porcine intestine, galanin is widely distributed in the central and peripheral nervous system. Galanin in most species is a 29 amino acid peptide with an amidated carboxyl terminus. Human galanin is unique in that it is longer, 30 amino acids, and is not amidated. There is strong conservation of the galanin sequence with the amino terminal fifteen residues being absolutely conserved in all species. Galanin immunoreactivity and binding is abundant in the hypothalamus, the locus coeruleus, the hippocampus and the anterior pituitary, as well as regions of the spinal cord, the pancreas and the gastrointestinal tract.

[0006] Like neuropeptide Y (NPY), injection of galanin into the paraventricular nucleus (PVN) of the hypothalamus produces a dose-dependent increase in feeding in satiated rats. While galanin, like norepinephrine, enhances carbohydrate ingestion, some studies have shown that it profoundly increases fat intake. It has been suggested that galanin shifts macronutrient preference from carbohydrate to fat. The same injections that increase feeding reduce energy expenditure and inhibit insulin secretion. There is enhanced galanin expression in the hypothalamus of genetically obese rats compared with their lean littermate controls. Injection of peptide receptor antagonists into the PVN blocks the galanin-specific induction of increased fat intake. Specific galanin antisense oligonucleotides when injected into the PVN produce a specific decrease in galanin expression associated with a decrease in fat ingestion and total caloric intake while hardly affecting either protein or carbohydrate intake. Thus galanin appears to be one potential neurochemical marker related to the behavior of fat ingestion.

[0007] Galanin inhibits cholinergic function and impairs working memory in rats. Lesions that destroy cholinergic neurons result in deficits in spatial learning tasks. While locally administered acetylcholine (ACh) reverses some of this deficit, galanin blocks this ACh-mediated improvement. Evidence from autopsy samples from Alzheimer's disease-afflicted brains suggests an increased galinergic innervation of the nucleus basilis. Thus, if galinergic overactivity contributes to the decline in cognitive performance in Alzheimer's disease, galanin antagonists may be therapeutically useful in alleviating cognitive impairment.

[0008] In the rat, administration of galanin intracerebroventricu-larly, subcutaneously or intravenously increases plasma growth hormone. Infusion of human galanin into healthy subjects also increases plasma growth hormone and potently enhances the growth hormone response to GHRH.

[0009] Galanin levels are particularly high in dorsal root ganglia. Sciatic nerve resection dramatically up-regulates galanin peptide and mRNA levels. Chronic administration of galanin receptor antagonists (M35, M15) after axotomy results in a marked increase in self mutilation behavior in rats, generally considered to be a response to pain. Application of antisense oligonucleotides specific for galanin to the proximal end of a transected sciatic nerve suppressed the increase in galanin peptide levels with a parallel increase in autotomy. Galanin injected intrathecally acts synergistically with morphine to produce analgesia, this antinociceptive effect of morphine is blocked by galanin receptor antagonists. Thus, galanin agonists may have some utility in relieving neural pain.

[0010] The actions of galanin are mediated by high affinity galanin receptors that are coupled by pertussis toxin sensitive G_(i)/G_(o) proteins to inhibition of adenylate cyclase activity, closure of L-type Ca⁺⁺ channels and opening of AT?-sensitive K⁺ channels. Specific binding of ¹²⁵I-galanin (Kd approximately 1 nM) has been demonstrated in areas paralleling localization of galanin immunoreactivity: hypothalamus, ventral hippocampus, basal forebrain, spinal cord, pancreas and pituitary. In most tissues the amino terminus (GAL 1-15) is sufficient for high affinity binding and agonist activity.

[0011] Recently, a galanin receptor cDNA was isolated by expression cloning from a human Bowes melanoma cell line. (Habert-Ortoli, et al. 1994. Proc. Nat. Acad. Sci., USA 91: 9780-9783). This receptor, GALR1, is expressed in human fetal brain and small intestine, but little else is known of its distribution. Gal(1-16) is at least 1000 times more active than pGAL(3-29) as an inhibitor of ¹²⁵I-porcine galanin binding to this receptor transiently expressed in COS cells. It remains to be determined whether this receptor subtype represents the hypothalamic receptor that mediates the galanin specific feeding behavior.

[0012] It would be desirable to identify further galanin receptors so that they can be used to further characterize this biological system and to identify galanin receptor subtype selective agonists and antagonists.

SUMMARY OF THE INVENTION

[0013] This invention relates to a novel galanin receptor, designated GALR3, substantially free from associated proteins, and to GALR3-like receptors which are at least about 40% homologous and which have substantially the same biological activity.

[0014] In preferred embodiments of this invention, the GALR3-like receptors are at least about 60%, and more preferably at least about 75%, and even more preferably at least about 85% homologous to a GALR3 receptor. This invention also relates specifically to rat, human and mouse GALR3, substantially free from associated proteins, and to receptors which are at least about 50% homologous and which have substantially the same biological activity.

[0015] Another aspect of this invention are primate and non-primate GALR3 proteins which are encoded by substantially the same nucleic acid sequences, but which have undergone changes in splicing or other RNA processing-derived modifications or mutagenesis-induced changes, so that the expressed protein has a homologous, but different amino acid sequence from the native forms. These variant forms may have different and/or additional functions in human and animal physiology or in vitro in cell based assays.

[0016] A further aspect of this invention are nucleic acids which encode a GALR3 receptor, a GALR3-like receptor or a functional equivalent of a GALR3 receptor from rat, human, mouse, swine, or other species. These nucleic acids may be free from associated nucleic acids, or they may be isolated or purified. The nucleic acids which encode a receptor of this invention may be any type of nucleic acid. Preferred forms are DNAs, including genomic and cDNA, although this invention specifically includes RNAs as well. Nucleic acid constructs may also contain regions which control transcription and translation such as one or more promoter regions, termination regions, and if desired enhancer regions. The nucleic acids may be inserted into any known vector including plasmids, and used to transfect suitable host cells using techniques generally available to one of ordinary skill in the art.

[0017] Another aspect of this invention are vectors comprising nucleic acids which encode GALR3, and host cells which contain these vectors. Still another aspect of this invention is a method of making GALR3 comprising introducing a vector comprising nucleic acids encoding GALR3 into a host cell under culturing conditions.

[0018] Yet another aspect of this invention are assays for GALR3 ligands which utilize the receptors and/or nucleic acids of this invention. Preferred assays of this embodiment compare the binding of the putative GALR3 ligand to the binding of galanin to GALR3.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 is the DNA sequence of human GALR3 gene, clone GALR3-3 (SEQ ID NO:1).

[0020]FIG. 2 is the deduced amino acid sequence of human GALR3, clone GALR3-3 (SEQ ID NO:2).

[0021]FIG. 3 is the DNA sequence (open reading frame only) of human GALR3, clone GALR3-2 (SEQ ID NO:3).

[0022]FIG. 4 is the deduced amino acid sequence of GALR3, clone GALR3-2 (SEQ ID NO:4)

[0023]FIG. 5 is a comparison of the open reading frame protein sequences of human and rat GALR3 with the corresponding sequences of GALR1 (mouse—SEQ ID NO:5, rat—SEQ ID NO:6, and human—SEQ ID NO:7) and GALR2 (mouse—SEQ ID NO:8, rat—SEQ ID NO:9, and human—SEQ ID NO:10).

[0024]FIG. 6 is a phylogenetic analysis of the putative GALR3 protein sequence.

[0025]FIG. 7 illustrates the competition curves for ¹²⁵I-porcine galanin against human and porcine galanin.

[0026]FIG. 8 is the DNA sequence of rat GALR3 from region TM4 to region TM7 (SEQ ID NO:11).

[0027]FIG. 9 is the deduced amino acid sequence of rat GALR3 from region TM4 to region TM7 (SEQ ID NO:12).

DETAILED DESCRIPTION OF THE INVENTION

[0028] As used throughout the specification and claims, the following definitions apply:

[0029] “Substantially free from associated proteins” means that the receptor is at least about 90%, and preferably at least about 95% free from other cell membrane proteins which are normally found in a living mammalian cell which expresses a galanin receptor.

[0030] “Substantially free from associated nucleic acids” means that the nucleic acid is at least about 90%, and preferably at least about 95%, free from other nucleic acids which are normally found in a living mammalian cell which naturally expresses a galanin receptor gene.

[0031] “Substantially the same biological activity” means that the receptor-galanin binding constant is within 5-fold of the binding constant of GALR3 and galanin, and preferably within 2-fold of the binding constant of GALR3 and galanin.

[0032] “Stringent post-hybridizational washing conditions” means 0.1× standard saline citrate (SSC) at 65° C.

[0033] “Standard post-hybridizational washing conditions” means 6× SSC at 55° C.

[0034] “Relaxed post-hybridizational washing conditions” means 6× SSC at 30° C., or 1 to 2× SSC at 55° C.

[0035] “Functional equivalent” means that a receptor which does not have the exact same amino acid sequence of a naturally occurring GALR3 protein due to alternative splicing, deletions, mutations, or additions, but retains at least 1%, preferably 10%, and more preferably 25% of the biological activity of the naturally occurring receptor. Such derivatives will have a significant homology with a natural GALR3 and can be detected by reduced stringency hybridization with a DNA sequence obtained from a GALR3. The nucleic acid coding a functional equivalent has at least about 60% homology at the nucleotide level to a naturally occurring receptor nucleic acid.

[0036] It has been found, in accordance with this invention, that there is a third galanin receptor, which is designated GALR3. The human (clone 3-3 and 3-2) and rat GALR3 sequences are given in FIGS. 1, 3 and 8, respectively, and are referenced in the Examples; however it is to be understood that this invention specifically includes GALR3 without regard to the species and, in particular, specifically includes rodent (including rat and mouse), rhesus, swine, chicken, cow and human. The galanin 3 receptors are highly conserved throughout species, and one of ordinary skill in the art, given the rat, human and/or mouse sequences presented herein, can easily design probes to obtain the GALR3 from other species.

[0037] GALR3 proteins contain various functional domains, including one or more domains which anchor the receptor in the cell membrane, and at least one ligand binding domain. As with many receptor proteins, it is possible to modify many of the amino acids, particularly those which are not found in the ligand binding domain, and still retain at least a percentage of the biological activity of the original receptor. Thus this invention specifically includes modified functionally equivalent GALR3s which have deleted, truncated, or mutated N-terminal portions. This invention also specifically includes modified functionally equivalent GALR3s which contain modifications and/or deletions in other domains, which are not accompanied by a loss of functional activity.

[0038] Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention.

[0039] The proteins of this invention were found to have structural features which are typical of the 7-transmembrane domain (TM) containing G-protein linked receptor superfamily (GPC-R's or 7-TM receptors). Thus GALR3 proteins make up new members of the GPC-R family of receptors. The intact GALR3 of this invention was found to have the general features of GPC-R's, including seven transmembrane regions, three intra- and extracellular loops, and the GPC-R protein signature sequence. The TM domains and GPC-R protein signature sequence are noted in the protein sequences of the GALR3. Not all regions are required for functioning, and therefore this invention also comprises functional receptors which lack one or more non-essential domains.

[0040] Determination of the nucleotide sequence indicated that the GALR3 belongs to the intron-containing class of GPC-R's.

[0041] The DNA sequence encoding the putative GALR3 is shown in FIGS. 1 and 3. The human putative GALR3 gene is organized similarly to human GALR2 with a single intron (−1 kb) dividing the open reading into two exons with Exon 1 consisting of −350 bp, and Exon 2-700 bp. Based on database searching, the open reading frame protein sequence for this novel gene (FIGS. 2 and 4) is most closely related to GALR2 and GALR1 with 58, 75% identity and similarity to human GALR2, and 37, 61% identity and similarity to rat GALR1 (FIG. 5). Differences in open reading frame DNA sequence and the resulting deduced amino acid sequence between clone GALR3-2 and GALR3-3 may be allelic in nature. Phylogenetic analysis of the putative GALR3 protein sequence supports the notion that this gene encodes a receptor for galanin (FIG. 6).

[0042] The human GALR3 protein bears strong sequence identity and similarity to the rat GALR3 ortholog.

[0043] This invention also relates to truncated forms of GALR3, particularly those which encompass the extracellular portion of the receptor, but lack the intracellular signaling portion of the receptor, and to nucleic acids encoding these truncated forms. Such truncated receptors are useful in various binding assays. Thus this invention specifically includes modified functionally equivalent GALR3s which have deleted, truncated, or mutated N-terminal portions. This invention also specifically includes modified functionally equivalent GALR3s including receptor chimeras which contain modifications and/or deletions in other domains, which are not accompanied by a loss of functional activity.

[0044] Additionally, it is possible to modify other functional domains such as those that interact with second messenger effector systems, by altering binding specificity and/or selectivity. Such functionally equivalent mutant receptors are also within the scope of this invention.

[0045] Assays which make up further aspects of this invention include binding assays (competition for ¹²⁵I-galanin binding), coupling assays (including galanin-mediated inhibition of forskolin-stimulated adenylate cyclase in cells expressing galanin receptors), measurement of galanin-stimulated calcium release in cells expressing galanin receptors (such as aequorin assays), stimulation of inward rectifying potassium channels (GIRK channels, measured by voltage changes) in cells expressing galanin receptors, and measurement of pH changes upon galanin stimulation of cells expressing galanin receptors as measured with a microphysiometer.

[0046] Host cells may be cultured under suitable conditions to produce GALR3. An expression vector containing DNA encoding the receptor may be used for expression of receptor in a recombinant host cell. Recombinant host cells may be prokaryotic or eukaryotic, including but not limited to bacteria such as E. coli, fungal cells such as yeast, mammalian cells including but not limited to cell lines of human, bovine, porcine, monkey and rodent origin, and insect cells including but not limited to Drosophila, Spodoptera, and silkworm derived cell lines. Cell lines derived from mammalian species which are suitable and which are commercially available include, but are not limited to, L cells L-M(TK⁻) (ATCC CCL 1.3), L cells L-M (ATCC CCL 1.2), 293 (ATCC CRL 1573), Raji (ATCC CCL 86), CV-1 (ATCC CCL 70), COS-1 (ATCC CRL 1650), COS-7 (ATCC CRL 1651), CHO-KI (ATCC CCL 61), 3T3 (ATCC CCL 92), NIH/3T3 (ATCC CRL 1658), HeLa (ATCC CCL 2), C127I (ATCC CRL 1616), BS-C-1 (ATCC CCL 26) and MRC-5 (ATCC CCL 171).

[0047] The specificity of binding of compounds showing affinity for the receptor is shown by measuring the affinity of the compounds for cells transfected with the cloned receptor or for membranes from these cells. Expression of the cloned receptor and screening for compounds that inhibit the binding of radiolabeled ligand to these cells provides a rational way for rapid selection of compounds with high affinity for the receptor. These compounds identified by the above assays may be agonists or antagonists of the receptor and may be peptides, proteins, or non-proteinaceous organic molecules. Alternatively, functional assays of the receptor may be used to screen for compounds which affect the activity of the receptor. Such functional assays range from ex vivo muscle contraction assays to assays which determine second messenger levels in cells expressing the receptor. The second messenger assays include, but are not limited to, assays to measure cyclic AMP or calcium levels or assays to measure adenyl cyclase activity. These compounds identified by the above assays may be agonists, antagonists, suppressors, or inducers of the receptor. The functional activity of these compounds is best assessed by using the receptor either natively expressed in tissues or cloned and exogenously expressed.

[0048] Using the assays of this invention, galanin agonists and antagonists may be identified. A galanin agonist is a compound which binds to the GALR3, such as a galanin mimetic, and produces a cellular response which is at least about equivalent to that of galanin, and which may be greater than that of galanin. Such compounds would be useful in situations where galanin insufficiency causes anorexia, or for treatment of pain.

[0049] Also using this embodiment of the assay, galanin antagonists may be identified. A galanin antagonist is a compound which can bind to the GALR3, but produces a lesser response than that of native galanin. Such compounds would be useful in the treatment of obesity.

[0050] One assay of this invention is a method of identifying a compound which modulates GALR3 receptor comprising: a) culturing cells expressing the GALR3 receptor in the presence of the compound and b) measuring GALR3 receptor activity or second messenger activity. If desired, the determined activity can be compared to a standard, such as that measured using galanin as the compound. In preferred embodiments, the cells are transformed and express the GALR3 receptor.

[0051] The consultant cDNA clone (or shorter portions of, for instance, only 15 nucleotides long) may be used to probe libraries under hybridization conditions to find other receptors which are similar enough so that the nucleic acids can hybridize, and is particularly useful for screening libraries from other species. In this step, one of ordinary skill in the art will appreciate that the hybridization conditions can vary from very stringent to relaxed. Proper temperature, salt concentrations, and buffers are well known.

[0052] The following non-limiting Examples are presented to better illustrate the invention.

EXAMPLE 1

[0053] Human GALR3:

[0054] Identification and Cloning of Human GalR3 Gene, Sequence and Gene Structure

[0055] Automated searching of sequence data from GenBank (National Center For Biotechnology Information, Bethesda, Md.) were queried using sequences from known receptor clones. Using a list of 50-60 rhodopsin family amino acid sequences, the NEW division of GenBank was searched. The query algorithm is TFASTX and the output is placed in a file where alignments are sorted by query sequence and scored (cut-off based on the expectation value, set for example, at 0.01). A DNA sequence alignment of 300 bp to a portion of a large BAC clone (-100,000 bp) with accession number Z97630 was identified from the high through-put genomic sequence (HTGS database, GenBank). The complete open reading frame (ORF) for the putative gene encoding GALR3 was then identified using sequence from BAC Z97630 and an additional BAC clone, with assession number Z82241, from the HTGS database. The Genbank assession numbers corresponded to the following HTGS BAC clones (HS entries): Z97630, HS466N1; Z82241, HS8112.

[0056] DNA sequences derived from these BACs were used to choose PCR primers. PCR primers beginning at the predicted initiating Met and ending more 3′ than the predicted stop codon were utilized to PCR from human genomic DNA a fragment containing the predicted Exon I, the intervening intron, and predicted Exon II. This PCR product was subcloned and sequenced, resulting in expression plasmid GALR3-3.

[0057] In a parallel approach, a human genomic DNA library (Stratagene, La Jolla, Calif.) was screened to isolate the putative GALR3 gene. Primary screening under medium stringency resulted in 6 positive plaques using an Exon 2 probe. One hybridizing phage plaque was obtained upon secondary screening. A 13 kb EcoR1/EcoRV fragment was identified from the genomic clone by Southern blotting, transferred into pBluescript vector (Stratagene, La Jolla, Calif.), and confirmed to be GALR3 by sequencing. A intronless GALR3 expression construct was assembled in a similar manner to that described above using Pfu DNA polymerase (Stratagene, La Jolla, Calif.) resulting in expression plasmid GALR3-2.

EXAMPLE 2

[0058] Chromosomal Location

[0059] The BAC clones which were identified by the searches of the HTGS dataset have been mapped by the The Sanger Centre (Cambridge, UK) genome research laboratory to human chromosome 22.

[0060] FISH analysis conducted herein has confirmed this assignment and refined it to 22q12.2-13.1.

EXAMPLE 3

[0061] Receptor Expression:

[0062] Construction of Human GalR3 Expression Plasmid

[0063] The human GalR3 cDNA expression construct was assembled stepwise from PCR products amplified from human genomic DNA. Each exon was PCR amplified using standard conditions. The primers in for exon I were: Forward Exon I (5′-gcg aat tcg gta cca tgg ctg atg ccc aga aca t-3′; SEQ ID NO:13) and Reverse Exon 1 (5′-cgc ctg tcg aca gat aca gca gc-3′; SEQ-ID NO:14). The primers for exon U were: Forward Exon II (5′-tgt atc tgt cga cag gta acc tgg ccg tgc ggc acc c-3′; SEQ ID NO: 15) and Reverse Exon II (5′-gcg cgg ccg ctt att ccg gtc ctc ggg c-3′; SEQ ID NO: 16). PCR products were subcloned into pCRII and sequenced. For expression in mammalian cells, the putative GALR3 ORF was subcloned into pcDNA-1/amp (Invitrogen) resulting in plasmid GALR3-3; and pcDNA-3 (Invitrogen), resulting in plasmid GALR3-2.

EXAMPLE 4

[0064] RNA Expression Profile

[0065] Using RNase protection analysis, the relative levels of human GALR3 mRNA was assessed. As shown below GALR3 is expressed in numerous brain regions and peripheral tissues, as observed for GALR1 and GALR2. Expression Tissue Level Amygdala + Cerebellum + Frontal Cortex + Hippocampus + Hypothalamus ++ Pituitary + Brain stem + Lung ++ Heart + Spleen + Liver + Pancreas + Duodenum + Colon + Straited muscle ++

EXAMPLE 5

[0066] Radioligand Binding:

[0067] Pharmacology of Human GALR3

[0068] Mammalian COS-7 cells were transfected by electroporation. COS-7 cells (1×10⁷) were suspended in 0.85 ml of Ringers' buffer and 15 mg of the GALR3-2 or GALR3-3 expression plasmid was added to a 0.4 mm electroporation cuvette (Bio-Rad, Hercules, Calif.). Current was applied (960 mF, 260 V) using a Bio-Rad Electroporator device and the cells were transferred to a T-180 flask (Corning). Expression was allowed to proceed for 72 hrs.

[0069] Membranes were prepared from transfected cells following dissociation in enzyme-free dissociation solution (Specialty Media, Lavallette, N.J.) by disruption in a Dounce homogenizer in ice-cold membrane buffer (10 mM Tris, pH 7.4, 10 mM PMSF, 10 mM phosphoramidon, and 40 mg/ml bacitracin). After a low speed (1100× g for 10 min. at 4° C.) and a high speed centrifugation (38,700× g for 15 min. at 4° C.), membranes were resuspended in buffer and protein concentration determined (Bio-Rad assay kit). Binding of ¹²⁵I-human or porcine galanin (specific activity of 2200 Ci/mmol, DuPont NEN) was measured in membranes using a buffer of 25 mM Tris pH 7.4, 0.5% BSA, 2 mM MgCl₂, 40 μg/ml bacitracin, 4 mg/ml phosphoramidon, and 10 μM leupeptin in a total volume of 250 ml. 70 pM ¹²⁵I-human or porcine galanin was used. Transfected cells expressing plasmid GALR3-3 were bound with ¹²⁵I-human galanin whereas cells expressing plasmid GALR3-2 were bound with ¹²⁵I-porcine galanin. Reactions were initiated by the addition of membranes and the incubation was allowed to proceed at room temperature for 1 hour. Non-specific binding was defined as the amount of radioactivity remaining bound in the presence of 1 mM respectively unlabeled galanin and was generally not above 200 cpm (<10% of total radioactivity bound). Titration of membrane protein from 1 to 50 μg was conducted. In competition studies various concentrations of unlabeled human or porcine galanin were included along with ¹²⁵I-porcine galanin (70 pM) in cells expressing the GALR3-2 plasmid. Incubations were terminated by rapid filtration through GF/C filters which had been presoaked with 0.1% polyethylamine using a TOMTEC (Orange, Conn.) cell harvester. The results were analyzed using the Prism software package (GraphPad, San Diego, Calif.). The table below illustrates that both clones confer specific binding to COS-7 cells for both human and porcine galanin radioligands as a function of protein concentration. In COS-7 cells mock transfected with expression vector only (no GALR3 gene), no specific binding of either radioligand was observed. Clone GALR3-3 Clone GALR3-2: Membrane Protein ¹²⁵I-human galanin ¹²⁵I-porcine galanin (μg) (cpm) (cpm) 1 ND ND 5 211 695 10 407 1134 20 886 1763 50 2061 3728

[0070] Competition curves for ¹²⁵I-porcine galanin against human and porcine galanin were generated to to determine the IC50 for both unlabeled peptides (clone GALR3-2), as shown in FIG. 7. The IC50 values for porcine and human galanin were 16 nM and 93 nM, respectively.

EXAMPLE 6

[0071] Rat GALR3:

[0072] Identification and Cloning of Rat GalR3 Gene

[0073] Primers based on the intronless human GALR3 sequence from TM4 and TM7 were designed and used to PCR amplify with Pfu DNA polymerase the rat GALR3 ortholog from rat genomic DNA. A PCR product of the appropriate size (approximately 400 bp) that hybridized with an Exon 2 probe from the human GALR3 gene was subcloned into pBluescript vector (Stratagene, La Jolla, Calif.). The DNA sequence is shown is FIG. 8 and the deduced amino acid sequence is shown in FIG. 9. DNA sequence analysis revealed significant homology with human GALR3: approximately 95% protein sequence identity for 129 amino acids spanning TM4 through TM-7.

[0074] RNA Expression

[0075] Northern blot analysis using a probe dervived from the rat GALR3 ORF has revealed expression of rat GALR3 mRNA in hypothalamus, mid-brain, pons, and whole fetal brain.

1 16 1 2263 DNA human 1 ccaggtcggg ggagttagat cccggggtca agcaaccaga actgggggct cttgcctgag 60 gattccagct tctcttccca ggtgcccgtc tgatggggag atggctgatg cccagaacat 120 ttcactggac agcccaggga gtgtgggggc cgtggcagtg cctgtggtct ttgccctaat 180 cttcctgctg ggcacagtgg gcaatgggct ggtgctggca gtgctcctgc agcctggccc 240 gagtgcctgg caggagcctc gcagcaccac ggacctgttc atcctcaacc tggcggtggc 300 tgacctctgc ttcatcctgt gctgcgtgcc cttccaggcc accatataca cgctggatgc 360 ctggctcttt ggggccctcg tttgcaaggc cgtgcacctg ttcatctacc tcaccatgaa 420 cgccagcagc tttacgctgg ctgctgtatc tgtggacagg tgcgctgtgc ctggggcctg 480 gctgggcagg gctgtggggg cgggggttgg gggaggagtc ctgaacagat cctcactggc 540 cttaggaagg agagagtggg ggaccagaaa gggaggtggg tgggaggaaa caaaagctcc 600 ctgacccctc gcaagcagcc tctgggcacc tgcagggcgt gcttgagggg actgtcctgc 660 ccttcccctc ctccactgtg aacttccaga ggacgcctct gagtctcaag tggcagcaca 720 gggtctggca catagtaagt gctctgtaag cgcgaaatga atcgcaaaag aagctcacga 780 atgcgttcat cagttttttt gttttgtttt gttttgttgt tttttttttt ttggatcttg 840 gctcactgca acctctgcct cctgggttcc agcgattctc ctgccacagc ctcctgagta 900 gctgggatta caggccacca cacctggcta attttttgta tttttagtag aaacggggtt 960 ttgccatgtt ggacaggctg gtctcgaacc cctgacctca agtgatccgc ccgcctcggc 1020 ctcccaagtg ctgggattac aggcgtgagc caccgcgccc agcccagcta ttttctaact 1080 gcccacacct ggccaagctg tgcacacatc tgcttccaca gcttgaaact tggggtcaaa 1140 tccaggctca ctccagctga tgaccctggg caagtcactt ctctctggac ctcatctgac 1200 gcatccataa aataatccta gaaataacaa gtcaccggga tcgggccctt gctaggtgca 1260 agggcctaag caccttgcgc gttcacaccc ttaatccccg ccacgtcccc cacggttcac 1320 aggaggcgca ctgggccgca gggcccgggc gcgggacgtg gcgcgggccc ctgcgggagg 1380 gcacctgccc gccccgctga ccasgcgccc tccgcaggta cctggccgtg cggcacccgc 1440 tgcgctcgcg cgccctgcgc acgccgcgta acgcccgcgc cgcagtgggg ctggtgtggc 1500 tgctggcggc gctcttctcg gcgccctacc tcagctacta cggcaccgtg cgctacggcg 1560 cgctggagct ctgcgtgccc gcctgggagg acgcgcgccg ccgcgccctg gacgtggcca 1620 ccttcgctgc cggctacctg ctgcccgtgg cygtggtgag cctggcctac gggcgcacgc 1680 tgcgcttcct gtgggccgcc gtgggtcccg cgggcgcggc ggcrgccaar gcgcggcgga 1740 gggcgackgg ccgcgcgggg cgcgccatgc tggcggtggc cgcgctctac gcgctmtgct 1800 ggggtccgca ccacgcgctc atcctgtgct tctggtacgg ccgmttcgcc ttcagcccgg 1860 ccacctacgc mtgccgcctg gcctcacact gcctggccta cgccaactcm tgcctcaacc 1920 cgctcgtmta cgcgctcgcc tcgcgccact tccgcgcgcg cttccgccgc ctgtggccgt 1980 gcggycgccg acgccgccac cgtgcccgcc gcgccttgcg tcgcgtccgc cccgcgtcct 2040 cgggcccacc cggctgcccc ggagacgccc ggcctagcgg gacgctgctg gctggtggcg 2100 gccagggccc sgagcccagg gagggacccg tccacggcgg agaggctgcc cgaggaccgg 2160 aataaaccct gccgcctgga ctccgcctgt gtccgtctgt ctcactcccg ttctccgaag 2220 gcgggacgcc accgggccag ggatggggca atgccacgag ctc 2263 2 368 PRT human 2 Met Ala Asp Ala Gln Asn Ile Ser Leu Asp Ser Pro Gly Ser Val Gly 1 5 10 15 Ala Val Ala Val Pro Val Val Phe Ala Leu Ile Phe Leu Leu Gly Thr 20 25 30 Val Gly Asn Gly Leu Val Leu Ala Val Leu Leu Gln Pro Gly Pro Ser 35 40 45 Ala Trp Gln Glu Pro Arg Ser Thr Thr Asp Leu Phe Ile Leu Asn Leu 50 55 60 Ala Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe Gln Ala 65 70 75 80 Thr Ile Tyr Thr Leu Asp Ala Trp Leu Phe Gly Ala Leu Val Cys Lys 85 90 95 Ala Val His Leu Phe Ile Tyr Leu Thr Met Asn Ala Ser Ser Phe Thr 100 105 110 Leu Ala Ala Val Ser Val Asp Arg Tyr Leu Ala Val Arg His Pro Leu 115 120 125 Arg Ser Arg Ala Leu Arg Thr Pro Arg Asn Ala Arg Ala Ala Val Gly 130 135 140 Leu Val Trp Leu Leu Ala Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr 145 150 155 160 Tyr Gly Thr Val Arg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala Trp 165 170 175 Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala Thr Phe Ala Ala Gly 180 185 190 Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala Tyr Gly Arg Thr Leu 195 200 205 Arg Phe Leu Trp Ala Ala Val Gly Pro Ala Gly Ala Ala Ala Ala Lys 210 215 220 Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg Ala Met Leu Ala Val 225 230 235 240 Ala Ala Leu Tyr Ala Leu Cys Trp Gly Pro His His Ala Leu Ile Leu 245 250 255 Cys Phe Trp Tyr Gly Arg Phe Ala Phe Ser Pro Ala Thr Tyr Ala Cys 260 265 270 Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn Ser Cys Leu Asn Pro 275 280 285 Leu Val Tyr Ala Leu Ala Ser Arg His Phe Arg Ala Arg Phe Arg Arg 290 295 300 Leu Trp Pro Cys Gly Arg Arg Arg Arg His Arg Ala Arg Arg Ala Leu 305 310 315 320 Arg Arg Val Arg Pro Ala Ser Ser Gly Pro Pro Gly Cys Pro Gly Asp 325 330 335 Ala Arg Pro Ser Gly Thr Leu Leu Ala Gly Gly Gly Gln Gly Pro Glu 340 345 350 Pro Arg Glu Gly Pro Val His Gly Gly Glu Ala Ala Arg Gly Pro Glu 355 360 365 3 1107 DNA human 3 atggctgatg cccagaacat ttcactggac agcccaggga gtgtgggggc cgtggcagtg 60 cctgtggtct ttgccctaat cttcctgctg ggcacagtgg gcaatgggct ggtgctggca 120 gtgctcctgc agcctggccc gagtgcctgg caggagcctg gcagcaccac ggacctgttc 180 atcctcaacc tggcggtggc tgacctctgc ttcatcctgt gctgcgtgcc cttccaggcc 240 accatctaca cgctggatgc ctggctcttt ggggccctcg tctgcaaggc cgtgcacctg 300 ctcatctacc tcaccatgta cgccagcagc tttacgctgg ctgctgtctc cgtggacagg 360 tacctggccg tgcggcaccc gctgcgctcg cgcgccctgc gcacgccgcg taacgcccgc 420 gccgcagtgg ggctggtgtg gctgctggcg gcgctcttct cggcgcccta cctcagctac 480 tacggcaccg tgcgctacgg cgcgctggag ctctgcgtgc ccgcctggga ggacgcgcgc 540 cgccgcgccc tggacgtggc caccttcgct gccggctacc tgctgcccgt ggctgtggtg 600 agcctggcct acgggcgcac gctgcgcttc ctgtgggccg ccgtgggtcc cgcgggcgcg 660 gcggcggccg aggcgcggcg gagggcgacg ggccgcgcgg ggcgcgccat gctggcggtg 720 gccgcgctct acgcgctctg ctggggtccg caccacgcgc tcatcctgtg cttctggtac 780 ggccgcttcg ccttcagccc ggccacctac gcctgccgcc tggcctcaca ctgcctggcc 840 tacgccaact cctgcctcaa cccgctcgtc tacgcgctcg cctcgcgcca cttccgcgcg 900 cgcttccgcc gcctgtggcc gtgcggccgc cgacgccgcc accgtgcccg ccgcgccttg 960 cgtcgcgtcc gccccgcgtc ctcgggccca cccggctgcc ccggagacgc ccggcctagc 1020 gggaggctgc tggctggtgg cggccagggc ccggagccca gggagggacc cgtccacggc 1080 ggagaggctg cccgaggacc ggaataa 1107 4 368 PRT human 4 Met Ala Asp Ala Gln Asn Ile Ser Leu Asp Ser Pro Gly Ser Val Gly 1 5 10 15 Ala Val Ala Val Pro Val Val Phe Ala Leu Ile Phe Leu Leu Gly Thr 20 25 30 Val Gly Asn Gly Leu Val Leu Ala Val Leu Leu Gln Pro Gly Pro Ser 35 40 45 Ala Trp Gln Glu Pro Gly Ser Thr Thr Asp Leu Phe Ile Leu Asn Leu 50 55 60 Ala Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe Gln Ala 65 70 75 80 Thr Ile Tyr Thr Leu Asp Ala Trp Leu Phe Gly Ala Leu Val Cys Lys 85 90 95 Ala Val His Leu Leu Ile Tyr Leu Thr Met Tyr Ala Ser Ser Phe Thr 100 105 110 Leu Ala Ala Val Ser Val Asp Arg Tyr Leu Ala Val Arg His Pro Leu 115 120 125 Arg Ser Arg Ala Leu Arg Thr Pro Arg Asn Ala Arg Ala Ala Val Gly 130 135 140 Leu Val Trp Leu Leu Ala Ala Leu Phe Ser Ala Pro Tyr Leu Ser Tyr 145 150 155 160 Tyr Gly Thr Val Arg Tyr Gly Ala Leu Glu Leu Cys Val Pro Ala Trp 165 170 175 Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala Thr Phe Ala Ala Gly 180 185 190 Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala Tyr Gly Arg Thr Leu 195 200 205 Arg Phe Leu Trp Ala Ala Val Gly Pro Ala Gly Ala Ala Ala Ala Glu 210 215 220 Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg Ala Met Leu Ala Val 225 230 235 240 Ala Ala Leu Tyr Ala Leu Cys Trp Gly Pro His His Ala Leu Ile Leu 245 250 255 Cys Phe Trp Tyr Gly Arg Phe Ala Phe Ser Pro Ala Thr Tyr Ala Cys 260 265 270 Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn Ser Cys Leu Asn Pro 275 280 285 Leu Val Tyr Ala Leu Ala Ser Arg His Phe Arg Ala Arg Phe Arg Arg 290 295 300 Leu Trp Pro Cys Gly Arg Arg Arg Arg His Arg Ala Arg Arg Ala Leu 305 310 315 320 Arg Arg Val Arg Pro Ala Ser Ser Gly Pro Pro Gly Cys Pro Gly Asp 325 330 335 Ala Arg Pro Ser Gly Arg Leu Leu Ala Gly Gly Gly Gln Gly Pro Glu 340 345 350 Pro Arg Glu Gly Pro Val His Gly Gly Glu Ala Ala Arg Gly Pro Glu 355 360 365 5 348 PRT mouse 5 Met Glu Leu Ala Met Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15 Glu Pro Pro Ala Pro Glu Ser Arg Pro Leu Phe Gly Ile Gly Val Glu 20 25 30 Asn Phe Ile Thr Leu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val 35 40 45 Leu Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly 50 55 60 Lys Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala 65 70 75 80 Asp Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr 85 90 95 Ala Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His 100 105 110 Tyr Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala 115 120 125 Met Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser 130 135 140 Ser Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Phe Ile Trp 145 150 155 160 Ala Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr His Gln Arg Leu 165 170 175 Phe His Arg Asp Ser Asn Gln Thr Phe Cys Trp Glu Gln Trp Pro Asn 180 185 190 Lys Leu His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly Tyr 195 200 205 Leu Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu Asn 210 215 220 His Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys Ser Glu Ala Ser 225 230 235 240 Lys Lys Lys Thr Ala Gln Thr Val Leu Val Val Val Val Val Phe Gly 245 250 255 Ile Ser Trp Leu Pro His His Val Val His Leu Trp Ala Glu Phe Gly 260 265 270 Ala Phe Pro Leu Thr Pro Ala Ser Phe Phe Phe Arg Ile Thr Ala His 275 280 285 Cys Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe 290 295 300 Leu Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys His 305 310 315 320 Val Cys Asp Glu Ser Pro Arg Ser Glu Thr Lys Glu Asn Lys Ser Arg 325 330 335 Met Asp Thr Pro Pro Ser Thr Asn Cys Thr His Val 340 345 6 346 PRT rat 6 Met Glu Leu Ala Pro Val Asn Leu Ser Glu Gly Asn Gly Ser Asp Pro 1 5 10 15 Glu Pro Pro Ala Glu Pro Arg Pro Leu Phe Gly Ile Gly Val Glu Asn 20 25 30 Phe Ile Thr Leu Val Val Phe Gly Leu Ile Phe Ala Met Gly Val Leu 35 40 45 Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly Lys 50 55 60 Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala Asp 65 70 75 80 Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr Ala 85 90 95 Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His Tyr 100 105 110 Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala Met 115 120 125 Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser Ser 130 135 140 Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Phe Ile Trp Ala 145 150 155 160 Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr Tyr Gln Arg Leu Phe 165 170 175 His Arg Asp Ser Asn Gln Thr Phe Cys Trp Glu His Trp Pro Asn Gln 180 185 190 Leu His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly Tyr Leu 195 200 205 Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu Asn His 210 215 220 Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys Ser Glu Ala Ser Lys 225 230 235 240 Lys Lys Thr Ala Gln Thr Val Leu Val Val Val Val Val Phe Gly Ile 245 250 255 Ser Trp Leu Pro His His Val Ile His Leu Trp Ala Glu Phe Gly Ala 260 265 270 Phe Pro Leu Thr Pro Ala Ser Phe Phe Phe Arg Ile Thr Ala His Cys 275 280 285 Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala Phe Leu 290 295 300 Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys Arg Val 305 310 315 320 Cys Asn Glu Ser Pro His Gly Asp Ala Lys Glu Lys Asn Arg Ile Asp 325 330 335 Thr Pro Pro Ser Thr Asn Cys Thr His Val 340 345 7 349 PRT human 7 Met Glu Leu Ala Val Gly Asn Leu Ser Glu Gly Asn Ala Ser Cys Pro 1 5 10 15 Glu Pro Pro Ala Pro Glu Pro Gly Pro Leu Phe Gly Ile Gly Val Glu 20 25 30 Asn Phe Val Thr Leu Val Val Phe Gly Leu Ile Phe Ala Leu Gly Val 35 40 45 Leu Gly Asn Ser Leu Val Ile Thr Val Leu Ala Arg Ser Lys Pro Gly 50 55 60 Lys Pro Arg Ser Thr Thr Asn Leu Phe Ile Leu Asn Leu Ser Ile Ala 65 70 75 80 Asp Leu Ala Tyr Leu Leu Phe Cys Ile Pro Phe Gln Ala Thr Val Tyr 85 90 95 Ala Leu Pro Thr Trp Val Leu Gly Ala Phe Ile Cys Lys Phe Ile His 100 105 110 Tyr Phe Phe Thr Val Ser Met Leu Val Ser Ile Phe Thr Leu Ala Ala 115 120 125 Met Ser Val Asp Arg Tyr Val Ala Ile Val His Ser Arg Arg Ser Ser 130 135 140 Ser Leu Arg Val Ser Arg Asn Ala Leu Leu Gly Val Gly Cys Ile Trp 145 150 155 160 Ala Leu Ser Ile Ala Met Ala Ser Pro Val Ala Tyr His Gln Gly Leu 165 170 175 Phe His Pro Arg Ala Ser Asn Gln Thr Phe Cys Trp Glu Gln Trp Pro 180 185 190 Asp Pro Arg His Lys Lys Ala Tyr Val Val Cys Thr Phe Val Phe Gly 195 200 205 Tyr Leu Leu Pro Leu Leu Leu Ile Cys Phe Cys Tyr Ala Lys Val Leu 210 215 220 Asn His Leu His Lys Lys Leu Lys Asn Met Ser Lys Lys Ser Glu Ala 225 230 235 240 Ser Lys Lys Lys Thr Ala Gln Thr Val Leu Val Val Val Val Val Phe 245 250 255 Gly Ile Ser Trp Leu Pro His His Ile Ile His Leu Trp Ala Glu Phe 260 265 270 Gly Val Phe Pro Leu Thr Pro Ala Ser Phe Leu Phe Arg Ile Thr Ala 275 280 285 His Cys Leu Ala Tyr Ser Asn Ser Ser Val Asn Pro Ile Ile Tyr Ala 290 295 300 Phe Leu Ser Glu Asn Phe Arg Lys Ala Tyr Lys Gln Val Phe Lys Cys 305 310 315 320 His Ile Arg Lys Asp Ser His Leu Ser Asp Thr Lys Glu Asn Lys Ser 325 330 335 Arg Ile Asp Thr Pro Pro Ser Thr Asn Cys Thr His Val 340 345 8 371 PRT mouse 8 Met Asn Gly Ser Asp Ser Gln Gly Ala Glu Asp Ser Ser Gln Glu Gly 1 5 10 15 Gly Gly Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu Phe Phe Ala 20 25 30 Leu Ile Phe Leu Val Gly Ala Val Gly Asn Ala Leu Val Leu Ala Val 35 40 45 Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile Leu 50 55 60 Asn Leu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro Phe 65 70 75 80 Gln Ala Thr Ile Tyr Thr Leu Asp Asp Trp Val Phe Gly Ser Leu Leu 85 90 95 Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser Ser 100 105 110 Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg Tyr 115 120 125 Pro Met His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala Ala 130 135 140 Ile Gly Leu Ile Trp Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr Leu 145 150 155 160 Ser Tyr Tyr Ser Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His Pro 165 170 175 Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp Leu Cys Thr Phe Val 180 185 190 Phe Ser Tyr Leu Leu Pro Val Leu Val Leu Ser Leu Thr Tyr Ala Arg 195 200 205 Thr Leu His Tyr Leu Trp Arg Thr Val Asp Pro Val Ala Ala Gly Ser 210 215 220 Gly Ser Gln Arg Ala Lys Arg Lys Val Thr Arg Met Ile Val Ile Val 225 230 235 240 Ala Val Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu Ile Leu 245 250 255 Cys Val Trp Phe Gly Arg Phe Pro Leu Thr Arg Ala Thr Tyr Ala Leu 260 265 270 Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn Pro 275 280 285 Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg Lys 290 295 300 Ile Cys Ala Gly Leu Leu Arg Arg Ala Pro Arg Arg Ala Ser Gly Arg 305 310 315 320 Val Cys Ile Leu Ala Pro Gly Asn His Ser Gly Gly Met Leu Glu Pro 325 330 335 Glu Ser Thr Asp Leu Thr Gln Val Ser Glu Ala Ala Gly Pro Leu Val 340 345 350 Pro Ala Pro Ala Leu Pro Asn Cys Thr Thr Leu Ser Arg Thr Leu Asp 355 360 365 Pro Ala Cys 370 9 372 PRT rat 9 Met Asn Gly Ser Gly Ser Gln Gly Ala Glu Asn Thr Ser Gln Glu Gly 1 5 10 15 Gly Ser Gly Gly Trp Gln Pro Glu Ala Val Leu Val Pro Leu Phe Phe 20 25 30 Ala Leu Ile Phe Leu Val Gly Thr Val Gly Asn Ala Leu Val Leu Ala 35 40 45 Val Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile 50 55 60 Leu Asn Leu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro 65 70 75 80 Phe Gln Ala Thr Ile Tyr Thr Leu Asp Asp Trp Val Phe Gly Ser Leu 85 90 95 Leu Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser 100 105 110 Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg 115 120 125 Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala 130 135 140 Ala Ile Gly Leu Ile Trp Gly Leu Ala Leu Leu Phe Ser Gly Pro Tyr 145 150 155 160 Leu Ser Tyr Tyr Arg Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His 165 170 175 Pro Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp Leu Cys Thr Phe 180 185 190 Val Phe Ser Tyr Leu Leu Pro Val Leu Val Leu Ser Leu Thr Tyr Ala 195 200 205 Arg Thr Leu Arg Tyr Leu Trp Arg Thr Val Asp Pro Val Thr Ala Gly 210 215 220 Ser Gly Ser Gln Arg Ala Lys Arg Lys Val Thr Arg Met Ile Ile Ile 225 230 235 240 Val Ala Val Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu Ile 245 250 255 Leu Cys Val Trp Phe Gly Arg Phe Pro Leu Thr Arg Ala Thr Tyr Ala 260 265 270 Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn 275 280 285 Pro Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg 290 295 300 Lys Ile Cys Ala Gly Leu Leu Arg Pro Ala Pro Arg Arg Ala Ser Gly 305 310 315 320 Arg Val Ser Ile Leu Ala Pro Gly Asn His Ser Gly Ser Met Leu Glu 325 330 335 Gln Glu Ser Thr Asp Leu Thr Gln Val Ser Glu Ala Ala Gly Pro Leu 340 345 350 Val Pro Pro Pro Ala Leu Pro Asn Cys Thr Ala Ser Ser Arg Thr Leu 355 360 365 Asp Pro Ala Cys 370 10 387 PRT human 10 Met Asn Val Ser Gly Cys Pro Gly Ala Gly Asn Ala Ser Gln Ala Gly 1 5 10 15 Gly Gly Gly Gly Trp His Pro Glu Ala Val Ile Val Pro Leu Leu Phe 20 25 30 Ala Leu Ile Phe Leu Val Gly Thr Val Gly Asn Thr Leu Val Leu Ala 35 40 45 Val Leu Leu Arg Gly Gly Gln Ala Val Ser Thr Thr Asn Leu Phe Ile 50 55 60 Leu Asn Leu Gly Val Ala Asp Leu Cys Phe Ile Leu Cys Cys Val Pro 65 70 75 80 Phe Gln Ala Thr Ile Tyr Thr Leu Asp Gly Trp Val Phe Gly Ser Leu 85 90 95 Leu Cys Lys Ala Val His Phe Leu Ile Phe Leu Thr Met His Ala Ser 100 105 110 Ser Phe Thr Leu Ala Ala Val Ser Leu Asp Arg Tyr Leu Ala Ile Arg 115 120 125 Tyr Pro Leu His Ser Arg Glu Leu Arg Thr Pro Arg Asn Ala Leu Ala 130 135 140 Ala Ile Gly Leu Ile Trp Gly Leu Ser Leu Leu Phe Ser Gly Pro Tyr 145 150 155 160 Leu Ser Tyr Tyr Arg Gln Ser Gln Leu Ala Asn Leu Thr Val Cys His 165 170 175 Pro Ala Trp Ser Ala Pro Arg Arg Arg Ala Met Asp Ile Cys Thr Phe 180 185 190 Val Phe Ser Tyr Leu Leu Pro Val Leu Val Leu Gly Leu Thr Tyr Ala 195 200 205 Arg Thr Leu Arg Tyr Leu Trp Arg Ala Val Asp Pro Val Ala Ala Gly 210 215 220 Ser Gly Ala Arg Arg Ala Lys Arg Lys Val Thr Arg Met Ile Leu Ile 225 230 235 240 Val Ala Ala Leu Phe Cys Leu Cys Trp Met Pro His His Ala Leu Ile 245 250 255 Leu Cys Val Trp Phe Gly Gln Phe Pro Leu Thr Arg Ala Thr Tyr Ala 260 265 270 Leu Arg Ile Leu Ser His Leu Val Ser Tyr Ala Asn Ser Cys Val Asn 275 280 285 Pro Ile Val Tyr Ala Leu Val Ser Lys His Phe Arg Lys Gly Phe Arg 290 295 300 Thr Ile Cys Ala Gly Leu Leu Gly Arg Ala Pro Gly Arg Ala Ser Gly 305 310 315 320 Arg Val Cys Ala Ala Ala Arg Gly Thr His Ser Gly Ser Val Leu Glu 325 330 335 Arg Glu Ser Ser Asp Leu Leu His Met Ser Glu Ala Ala Gly Ala Leu 340 345 350 Arg Pro Cys Pro Gly Ala Ser Gln Pro Cys Ile Leu Glu Pro Cys Pro 355 360 365 Gly Pro Ser Trp Gln Gly Pro Lys Ala Gly Asp Ser Ile Leu Thr Val 370 375 380 Asp Val Ala 385 11 390 DNA rat 11 ccctacctca gctactacgg cacggtgcgc tacggccggc tcgagctctg cgtgcccgct 60 tgggaggagg acgcgcggcg gcgcgcgctg gacgtggcca ccttcgccgc gggctacctg 120 ctgccggtgg ccgtggtgag cctggcctac ggacgcacgc tatgtttcct atgggccgcc 180 gtgggtcccg cgggcagcgc ggcagcagag gcgcgcagac gggcgaccgg ccgggcggga 240 cgccgcatgc tggcagtggc gctctacgcg ctttgctggg gcccgcacca cgcgctcatc 300 ctctgcttct ggtacggtcc gttcgccttc agcccggcca cctacgcctg tcgcctggcc 360 tcacactgcc tcgcctacgc caactcctgc 390 12 129 PRT rat 12 Pro Tyr Leu Ser Tyr Tyr Gly Thr Val Arg Tyr Gly Arg Leu Glu Leu 1 5 10 15 Cys Val Pro Ala Trp Glu Asp Ala Arg Arg Arg Ala Leu Asp Val Ala 20 25 30 Thr Phe Ala Ala Gly Tyr Leu Leu Pro Val Ala Val Val Ser Leu Ala 35 40 45 Tyr Gly Arg Thr Leu Cys Phe Leu Trp Ala Ala Val Gly Pro Ala Gly 50 55 60 Ser Ala Ala Ala Glu Ala Arg Arg Arg Ala Thr Gly Arg Ala Gly Arg 65 70 75 80 Arg Met Leu Ala Val Ala Leu Tyr Ala Leu Cys Trp Gly Pro His His 85 90 95 Ala Leu Ile Leu Cys Phe Trp Tyr Gly Pro Phe Ala Phe Ser Pro Ala 100 105 110 Thr Tyr Ala Cys Arg Leu Ala Ser His Cys Leu Ala Tyr Ala Asn Ser 115 120 125 Cys 13 34 DNA Artificial Sequence PCR primer 13 gcgaattcgg taccatggct gatgcccaga acat 34 14 23 DNA Artificial Sequence PCR primer 14 cgcctgtcga cagatacagc agc 23 15 37 DNA Artificial Sequence PCR primer 15 tgtatctgtc gacaggtaac ctggccgtgc ggcaccc 37 16 28 DNA Artificial Sequence PCR primer 16 gcgcggccgc ttattccggt cctcgggc 28 

What is claimed:
 1. Galanin receptor 3 (GALR3), substantially free from associated proteins, or a GALR3-like receptor, wherein the GALR3-like receptor shares at least about 40% homology to GALR3 and has substantially the same biological activity.
 2. A GALR3-like receptor according to claim 1, which shares at least about 50% homology to a GALR3.
 3. A GALR3-like receptor according to claim 1, which shares at least about 75% homology to a GALR3.
 4. A GALR3-like receptor according to claim 1, which shares at least about 85% homology to a GALR3.
 5. A GALR3 in accordance with claim 1 which is human.
 6. A GALR3 in accordance with claim 1 which is rat.
 7. GALR3 according to claim 1 which is shown in FIG.
 1. 8. GALR3 according to claim 1 which is shown in FIG.
 3. 9. GALR3 according to claim 1 which is shown in FIG.
 8. 10. A nucleic acid, substantially free from associated nucleic acids, which encodes a GALR3 or a GALR3-like receptor which is at least about 40% homologous to GALR3 and which has substantially the same biological activity.
 11. A nucleic acid encoding a GALR3-like receptor according to claim 10, wherein the GALR3-like receptor shares at least about 50% homology to a GALR3.
 12. A nucleic acid encoding a GALR3-like receptor according to claim 10, wherein the GALR3-like receptor shares at least about 75% homology to a GALR3.
 13. A nucleic acid encoding a GALR3-like receptor according to claim 10, wherein the GALR3-like receptor shares at least about 85% homology to human GALR3.
 14. A nucleic acid according to claim 10 which is DNA.
 15. A vector comprising the nucleic acid of claim
 13. 16. A host cell comprising the nucleic acid of claim
 10. 17. A method of determining if a compound is a GALR3 ligand comprising contacting the compound and GALR3 and determining if binding occurs.
 18. A method of identifying a compound that modulates GALR3 receptor activity, comprising: (a) culturing cells expressing GALR3 receptor in the presence of the compound; and (b) measuring GALR3 receptor activity or second messenger activity.
 19. A method according to claim 18 wherein the cells are transformed to express a GALR3 receptor. 